Development of a Weight-Band Dosing Approach for Vosoritide in Children with Achondroplasia Using a Population Pharmacokinetic Model.

BACKGROUND AND OBJECTIVE: Vosoritide is a recently approved therapy for achondroplasia, the most common form of disproportionate short stature, that has been shown to be well tolerated and effective in increasing linear growth. This study aimed to develop a population pharmacokinetic (PPK) model to characterize pharmacokinetics (PK) of vosoritide and establish a weight-band dosing regimen. METHODS: A PPK model was developed using data from five clinical trials in children with achondroplasia (aged 0.95-15 years) who received daily per-kg doses of vosoritide. The model was used to simulate expected exposures in children with a refined weight-band dosing regimen. Simulated exposure was compared with the observed exposure from the pivotal clinical trial to evaluate appropriateness of the weight-band dosing regimen. RESULTS: A one-compartment model with a change-point first-order absorption and first-order elimination accurately described PK of vosoritide in children with achondroplasia. Body weight was found to be a predictor of vosoritide's clearance and volume of distribution. Additionally, it was observed that dosing solution concentration and duration of treatment influenced bioavailability. The weight-band dosing regimen resulted in simulated exposures that were within the range demonstrated to be well tolerated and effective in the pivotal clinical trial and showed improved consistency in drug exposure across the achondroplasia population. CONCLUSIONS: The weight-band dosing regimen reduced the number of recommended dose levels by body weight and is expected to simplify dosing for children with achondroplasia and their caregivers. CLINICAL TRIAL REGISTRATION: NCT02055157, NCT02724228, NCT03197766, NCT03424018, and NCT03583697.

A long-acting C-natriuretic peptide for achondroplasia.

The C-natriuretic peptide (CNP) analog vosoritide has recently been approved for treatment of achondroplasia in children. However, the regimen requires daily subcutaneous injections in pediatric patients over multiple years. The present work sought to develop a long-acting CNP that would provide efficacy equal to or greater than that of vosoritide but require less frequent injections. We used a technology for half-life extension, whereby a drug is attached to tetra-polyethylene glycol hydrogels (tetra-PEG) by ß-eliminative linkers that cleave at predetermined rates. These hydrogels-fabricated as uniform ∼60-µm microspheres-are injected subcutaneously, where they serve as a stationary depot to slowly release the drug into the systemic circulation. We prepared a highly active, stable CNP analog-[Gln6,14]CNP-38-composed of the 38 C-terminal amino acids of human CNP-53 containing Asn to Gln substitutions to preclude degradative deamidation. Two microsphere [Gln6,14]CNP-38 conjugates were prepared, with release rates designed to allow once-weekly and once-monthly administration. After subcutaneous injection of the conjugates in mice, [Gln6,14]CNP-38 was slowly released into the systemic circulation and showed biphasic elimination pharmacokinetics with terminal half-lives of ∼200 and ∼600 h. Both preparations increased growth of mice comparable to or exceeding that produced by daily vosoritide. Simulations of the pharmacokinetics in humans indicated that plasma [Gln6,14]CNP-38 levels should be maintained within a therapeutic window over weekly, biweekly, and likely, monthly dosing intervals. Compared with vosoritide, which requires ∼30 injections per month, microsphere [Gln6,14]CNP-38 conjugates-especially the biweekly and monthly dosing-could provide an alternative that would be well accepted by physicians, patients, and patient caregivers.

Effect of C-type natriuretic peptide on craniofacial skeletogenesis in mice during the pubertal growth spurt.

OBJECTIVE: This study aimed to determine the effect of C-type natriuretic peptide (CNP) overexpression on craniofacial growth during the pubertal growth period in mice. DESIGN: Six-week-old C57BL/6 mice were injected with pLIVE-Empty vectors (Control mice) and pLIVE-NPPC vectors (CNP mice) using the hydrodynamic method. Morphological analyses were performed at the age of 12 weeks. RESULTS: Micro-computed tomography (µCT) images showed significant (p < 0.05) hyperplasia in the maxilla along the sagittal plane (CNP mice: 13.754 mm, Control mice: 13.215 mm). Further, the images revealed significant bone overgrowth in the sagittal direction in the sphenoid (CNP mice: 6.936 mm, Control mice: 6.411 mm) and occipital (CNP mice: 4.051 mm, Control mice: 3.784 mm) bones in the CNP mice compared with that in the Control mice. Compared with SAP-Nppc-Tg mice in previous studies, although there was no effect on nose length and nasal bone length, the effect was sufficient to improve craniofacial hypogrowth. Furthermore, CNP promoted sagittal cranial growth by increasing the thickness of the spheno-occipital synchondrosis in organ cultures and nasal septal cartilage in micromass cultures, which were derived from 6-week-old mice. CONCLUSIONS: We have previously shown that the elevated blood levels of CNP from the neonatal period affect midfacial skeletogenesis by promoting endochondral ossification using mice (SAP-Nppc-Tg mice). The overexpression of CNP, even in 6-weeks-old mice, promoted growth in the sagittal direction within the maxillary region. These findings indicate the therapeutic potential of CNP for the treatment of midfacial hypoplasia during the pubertal growth spurt.

C-Type Natriuretic Peptide Analogue Therapy in Children with Achondroplasia.

BACKGROUND: Achondroplasia is a genetic disorder that inhibits endochondral ossification, resulting in disproportionate short stature and clinically significant medical complications. Vosoritide is a biologic analogue of C-type natriuretic peptide, a potent stimulator of endochondral ossification. METHODS: In a multinational, phase 2, dose-finding study and extension study, we evaluated the safety and side-effect profile of vosoritide in children (5 to 14 years of age) with achondroplasia. A total of 35 children were enrolled in four sequential cohorts to receive vosoritide at a once-daily subcutaneous dose of 2.5 µg per kilogram of body weight (8 patients in cohort 1), 7.5 µg per kilogram (8 patients in cohort 2), 15.0 µg per kilogram (10 patients in cohort 3), or 30.0 µg per kilogram (9 patients in cohort 4). After 6 months, the dose in cohort 1 was increased to 7.5 µg per kilogram and then to 15.0 µg per kilogram, and in cohort 2, the dose was increased to 15.0 µg per kilogram; the patients in cohorts 3 and 4 continued to receive their initial doses. At the time of data cutoff, the 24-month dose-finding study had been completed, and 30 patients had been enrolled in an ongoing long-term extension study; the median duration of follow-up across both studies was 42 months. RESULTS: During the treatment periods in the dose-finding and extension studies, adverse events occurred in 35 of 35 patients (100%), and serious adverse events occurred in 4 of 35 patients (11%). Therapy was discontinued in 6 patients (in 1 because of an adverse event). During the first 6 months of treatment, a dose-dependent increase in the annualized growth velocity was observed with vosoritide up to a dose of 15.0 µg per kilogram, and a sustained increase in the annualized growth velocity was observed at doses of 15.0 and 30.0 µg per kilogram for up to 42 months. CONCLUSIONS: In children with achondroplasia, once-daily subcutaneous administration of vosoritide was associated with a side-effect profile that appeared generally mild. Treatment resulted in a sustained increase in the annualized growth velocity for up to 42 months. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov numbers, NCT01603095, NCT02055157, and NCT02724228.).

C-type natriuretic peptide attenuates renal osteodystrophy through inhibition of FGF-23/MAPK signaling.

Renal osteodystrophy (ROD) occurs as early as chronic kidney disease (CKD) stage 2 and seems ubiquitous in almost all pediatric patients with CKD stage 5. Fibroblast growth factor (FGF)-23, a bone-derived endocrine regulator of phosphate homeostasis, is overexpressed in CKD and disturbs osteoblast differentiation and matrix mineralization. In contrast, C-type natriuretic peptide (CNP) acts as a potent positive regulator of bone growth. In the present study, we infused CNP into uremic rats and observed whether CNP could attenuate ROD through the inhibition of FGF-23 cascades. In uremic rats, CNP administration significantly alleviated renal dysfunction, calcium phosphate metabolic disorders, hypovitaminosis D, secondary hyperparathyroidism, the decrease in bone turnover markers and retarded bone pathological progression. More importantly, within FGF-23/mitogen-activated protein kinase (MAPK) signaling, the fibroblast growth factor receptor-1, Klotho and alternative (STAT-1/phospho-STAT-1) elements were upregulated by CNP, whereas FGF-23, RAF-1/phospho-RAF-1, and downstream (ERK/phospho-ERK and P38/phospho-P38) elements were paradoxically underexpressed in bone tissue. Therefore, CNP exerts a therapeutic effect on ROD through inhibition of FGF-23/MAPK signaling at the RAF-1 level.

Chronic treatment with C-type natriuretic peptide impacts differently in the aorta of normotensive and hypertensive rats.

The aim of this study was to determine whether exogenous administration of C-type natriuretic peptide (CNP) induces functional and morphological vascular changes in spontaneously hypertensive rats (SHR) compared with normotensive rats. Male 12-week-old normotensive Wistar and SHR were administered with saline (NaCl 0.9%) or CNP (0.75 µg/h/100 g) for 14 days (subcutaneous micro-osmotic pumps). Systolic blood pressure (SBP) was measured in awake animals and renal parameters were evaluated. After decapitation, the aorta was removed, and vascular morphology, profibrotic markers, and vascular reactivity were measured. In addition, nitric oxide (NO) system and oxidative stress were evaluated. After 14-days of treatment, CNP effectively reduced SBP in SHR without changes in renal function. CNP attenuated vascular remodeling in hypertensive rats, diminishing both profibrotic and pro-inflammatory cytokines. Also, CNP activated the vascular NO system and exerted an antioxidant effect in aortic tissue of both groups, diminishing superoxide production and thiobarbituric acid-reactive substances, and increasing glutathione content. These results show that chronic treatment with CNP attenuates the vascular damage development in a model of essential hypertension, inducing changes in fibrotic, inflammatory, oxidative, and NO pathways that could contribute to beneficial long-term effects on vascular morphology, extracellular matrix composition, and function. The knowledge of these effects of CNP could lead to improved therapeutic strategies to not only control BP but also reduce vascular damage, primarily responsible for the risk of cardiovascular events.

C-Type Natriuretic Peptide Dampens Fibroblast Growth Factor-23 Expression Through MAPK Signaling Pathway in Human Mesangial Cells.

C-type natriuretic peptide (CNP) is believed to be produced locally in the kidneys and possess several renoprotective properties. In contrast, fibroblast growth factor (FGF) -23 elevates in the early stage of chronic kidney disease and predicts its outcomes. Currently, several studies have demonstrated that CNP and FGF-23 act through a close pathway, and moreover, FGF-23/mitogen-activated protein kinase (MAPK) can be obviously suppressed by CNP. In the present study, human mesangial cells (MCs) were incubated in serum-containing medium in the absence or presence of CNP (0, 10 and 100 pM) for 24, 48 and 72 h, respectively. CNP administration significantly suppresses MCs proliferation in a time- and dose-dependent manner. As a down-stream signaling of CNP activation, the expressions of natriuretic peptide receptor (NPR)-B, cyclic guanosine monophosphate-dependent protein kinases II and NPR-C were obviously augmented, whereas neutral endopeptidase expression was significantly decreased after CNP treatment in MCs. FGF-23, FGF receptor-1 and RAF-1 experienced a pronounced down-regulation in MCs with different doses of CNP throughout the whole observational period. CNP may dampen FGF-23 expression via MAPK signaling pathway in MCs.

Effects of pre-incubation with C-type natriuretic peptide on nuclear maturation, mitochondrial behavior, and developmental competence of sheep oocytes.

In vitro produced mammalian embryos suffer from developmental failure, with a large proportion showing embryonic retardation, degradation, or apoptosis. This failure is due, in part, to incomplete oocyte cytoplasmic maturation. C-type natriuretic peptide (CNP) has been reported to act as a meiotic inhibitor. Here we explored the potential effects of CNP pre-treatment sheep oocytes on nuclear maturation, changes in mitochondrial behavior and developmental competence of in vitro fertilized embryos. Sheep cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries. Nuclear progression was assessed using DAPI chromatin staining, the expression of natriuretic peptide receptor 2 (NPR2) was evaluated by RT-qPCR, active mitochondria localization was assessed with a confocal laser scanning microscopy using MitoTracker Red, and the developmental competence of sheep oocytes subjected to one-step IVM or two-step IVM with or without CNP pretreatment was also investigated. Our results showed that 200 nM CNP could effectively maintain meiotic arrest of sheep COCs in vitro within 4 h. Furthermore, NPR2 mRNA was mainly expressed in cumulus cells. COCs pre-treated with 200 nM CNP for 4 h followed by 24 h IVM showed significantly higher (P < 0.05) cleavage rate and blastocyst rate after in vitro fertilization (IVF), and significantly lower (P < 0.05) proportion of DNA-fragmented nuclei in blastocysts when compared to the conventional 24 h IVM (standard IVM). Non-matured oocytes mainly displayed brilliant circumferential and fine diffuse distribution of mitochondria throughout the cytoplasm. By comparison, 200 nM CNP pre-treated COCs for 4 h led to cytoplasmic mitochondrial granule localization to the peripheral and perinuclear regions. Moreover, oocytes pre-treated with 200 nM CNP for 4 h followed by 24 h IVM, showed mitochondrial organization were similar to those of conventional 24 h matured oocytes in which mitochondria were aggregated more toward the cortical regions of the oocytes, but with larger clumps of stained mitochondria. These results indicate that CNP pre-treatment improves the quality and developmental competence of sheep oocytes and has great potential for facilitating in vitro embryo production.

Comparative Efficacy of Drugs for Preventing Acute Kidney Injury after Cardiac Surgery: A Network Meta-Analysis.

BACKGROUND: Acute kidney injury (AKI) occurs frequently after cardiac surgery and has been associated with increased hospital length of stay, mortality, and costs. OBJECTIVE: We aimed to evaluate the efficacy of pharmacologic strategies for preventing AKI after cardiac surgery. METHODS: We searched PubMed, Embase, the Cochrane Central Register of Controlled Trials (CENTRAL) up to 6 May 2017 and the reference lists of relevant articles about trials. The outcome was the occurrence of AKI. This is the first network meta-analysis of the different prevention strategies using Bayesian methodology. RESULTS: The study included 63 articles with 19,520 participants and evaluated the effect of ten pharmacologic strategies to prevent AKI in patients undergoing cardiac surgery. Compared with placebo, the odds ratio (OR) for the occurrence of AKI was 0.24 [95% confidence interval (CI) 0.16-0.34] with natriuretic peptide, 0.33 (95% CI 0.14-0.70) with fenoldopam, 0.54 (95% CI 0.31-0.84) with dexmedetomidine, 0.56 (95% CI 0.29-0.95) with low-dose erythropoietin, 0.63 (95% CI 0.43-0.88) with levosimendan, 0.76 (95% CI 0.52-1.10) with steroids, 0.83 (95% CI 0.48-1.40) with high-dose erythropoietin, 0.85 (95% CI 0.64-1.14) with N-acetylcysteine, 0.96 (95% CI 0.69-1.29) with sodium bicarbonate, and 1.05 (95% CI 0.70-1.41) with statins. The surface under the cumulative ranking curve probabilities indicated that natriuretic peptide was the best treatment therapy and that fenoldopam ranked second. CONCLUSIONS: Natriuretic peptide is probably the preferred pharmacologic strategy to prevent AKI in adult patients undergoing cardiac surgery, especially in those at high risk of AKI.

Design and evaluation of novel natriuretic peptide derivatives with improved pharmacokinetic and pharmacodynamic properties.

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor B (NPR-B), are potent positive regulators of endochondral bone growth, making the CNP pathway one of the most promising therapeutic targets for the treatment of growth failure. However, the administration of exogenous CNP is not fully effective, due to its rapid clearance in vivo. Modification of CNP to potentially druggable derivatives may result in increased resistance to proteolytic degradation, longer plasma half-life (T1/2), and better distribution to target tissues. In the present study, we designed and evaluated CNP/ghrelin chimeric peptides as novel CNP derivatives. We have previously reported that the ghrelin C-terminus increases peptide metabolic stability. Therefore, we combined the 17-membered, internal disulfide ring portion of CNP with the C-terminal portion of ghrelin. The resultant peptide displayed improved biokinetics compared to CNP, with increased metabolic stability and longer plasma T1/2. Repeated subcutaneous administration of the chimeric peptide to mice resulted in a significant acceleration in longitudinal growth, whereas CNP(1-22) did not. These results suggest that the ghrelin C-terminus improves the stability of CNP, and the chimeric peptide may be useful as a novel therapeutic agent for growth failure and short stature.

C-type natriuretic peptide suppresses mesangial proliferation and matrix expression via a MMPs/TIMPs-independent pathway in vitro.

C-type natriuretic peptide (CNP) acts mainly in a local, paracrine fashion to regulate vascular tone and cell proliferation. Although several in vivo studies have demonstrated that CNP exerts an inhibitory effect on mesangial matrix generation, a limited number of reports exist about the anti-extracellular matrix (ECM) accumulation effect of CNP and its underlying mechanisms in mesangial cells (MCs) in vitro. In this study, human MCs were incubated in serum-containing medium in the absence or presence of CNP (0, 10 and 100 pM) for 24, 48 and 72 h, respectively. CNP administration significantly suppresses MCs proliferation and collagen (Col)-IV expression in a time- and dose-dependent manner. In addition, the study presented herein was designed as a first demonstration of the regulative effects of CNP on the metabolisms of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in MCs in vitro, and found that: (1) CNP administration significantly decreased the secretion and expression of MMP-2 and MMP-9 in the cultured MCs; (2) the secretion and expression of TIMP-1 progressively elevated after treatment with CNP for 24 and 48 h, whereas declined at later time point; (3) CNP expression was negatively correlated with MMP-2 and MMP-9 expression; (4) the balance of MMPs/TIMPs was shifted toward the reduction in MMP-2 and MMP-9 activity and/or the increment in TIMP-1 expression, which could not account for the down-regulation of Col-IV expression in CNP-treated MCs. In conclusion, CNP suppresses mesangial proliferation and ECM expression via a MMPs/TIMPs-independent pathway in vitro.

C-type natriuretic peptide-modified lipid vesicles: fabrication and use for the treatment of brain glioma.

Chemotherapy of brain glioma faces a major obstacle owing to the inability of drug transport across the blood-brain barrier (BBB). Besides, neovasculatures in brain glioma site result in a rapid infiltration, making complete surgical removal virtually impossible. Herein, we reported a novel kind of C-type natriuretic peptide (CNP) modified vinorelbine lipid vesicles for transferring drug across the BBB, and for treating brain glioma along with disrupting neovasculatures. The studies were performed on brain glioma U87-MG cells in vitro and on glioma-bearing nude mice in vivo. The results showed that the CNP-modified vinorelbine lipid vesicles could transport vinorelbine across the BBB, kill the brain glioma, and destroy neovasculatures effectively. The above mechanisms could be associated with the following aspects, namely, long circulation in the blood; drug transport across the BBB via natriuretic peptide receptor B (NPRB)-mediated transcytosis; elimination of brain glioma cells and disruption of neovasculatures by targeting uptake and cytotoxic injury. Besides, CNP-modified vinorelbine lipid vesicles could induce apoptosis of the glioma cells. The mechanisms could be related to the activations of caspase 8, caspase 3, p53, and reactive oxygen species (ROS), and inhibition of survivin. Hence, CNP-modified lipid vesicles could be used as a carrier material for treating brain glioma and disabling glioma neovasculatures.

Dorsal root ganglion axon bifurcation tolerates increased cyclic GMP levels: the role of phosphodiesterase 2A and scavenger receptor Npr3.

A cyclic GMP (cGMP) signaling pathway, comprising C-type natriuretic peptide (CNP), its guanylate cyclase receptor Npr2, and cGMP-dependent protein kinase I, is critical for the bifurcation of dorsal root ganglion (DRG) and cranial sensory ganglion axons when entering the mouse spinal cord and the hindbrain respectively. However, the identity and functional relevance of phosphodiesterases (PDEs) that degrade cGMP in DRG neurons are not completely understood. Here, we asked whether regulation of the intracellular cGMP concentration by PDEs modulates the branching of sensory axons. Real-time imaging of cGMP with a genetically encoded fluorescent cGMP sensor, RT-PCR screens, in situ hybridization, and immunohistology combined with the analysis of mutant mice identified PDE2A as the major enzyme for the degradation of CNP-induced cGMP in embryonic DRG neurons. Tracking of PDE2A-deficient DRG sensory axons in conjunction with cGMP measurements indicated that axon bifurcation tolerates increased cGMP concentrations. As we found that the natriuretic peptide scavenger receptor Npr3 is expressed by cells associated with dorsal roots but not in DRG neurons itself at early developmental stages, we analyzed axonal branching in the absence of Npr3. In Npr3-deficient mice, the majority of sensory axons showed normal bifurcation, but a small population of axons (13%) was unable to form T-like branches and generated turns in rostral or caudal directions only. Taken together, this study shows that sensory axon bifurcation is insensitive to increases of CNP-induced cGMP levels and Npr3 does not have an important scavenging function in this axonal system.

C-type natriuretic peptide ameliorates pulmonary fibrosis by acting on lung fibroblasts in mice.

BACKGROUND: Pulmonary fibrosis has high rates of mortality and morbidity; however, no effective pharmacological therapy has been established. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, selectively binds to the transmembrane guanylyl cyclase (GC)-B receptor and exerts anti-inflammatory and anti-fibrotic effects in various organs through vascular endothelial cells and fibroblasts that have a cell-surface GC-B receptor. Given the pathophysiological importance of fibroblast activation in pulmonary fibrosis, we hypothesized that the anti-fibrotic and anti-inflammatory effects of exogenous CNP against bleomycin (BLM)-induced pulmonary fibrosis were exerted in part by the effect of CNP on pulmonary fibroblasts. METHODS: C57BL/6 mice were divided into two groups, CNP-treated (2.5 µg/kg/min) and vehicle, to evaluate BLM-induced (1 mg/kg) pulmonary fibrosis and inflammation. A periostin-CNP transgenic mouse model exhibiting CNP overexpression in fibroblasts was generated and examined for the anti-inflammatory and anti-fibrotic effects of CNP via fibroblasts in vivo. Additionally, we assessed CNP attenuation of TGF-ß-induced differentiation into myofibroblasts by using immortalized human lung fibroblasts stably expressing GC-B receptors. Furthermore, to investigate whether CNP acts on human lung fibroblasts in a clinical setting, we obtained primary-cultured fibroblasts from surgically resected lungs of patients with lung cancer and analyzed levels of GC-B mRNA transcription. RESULTS: CNP reduced mRNA levels of the profibrotic cytokines interleukin (IL)-1ß and IL-6, as well as collagen deposition and the fibrotic area in lungs of mice with bleomycin-induced pulmonary fibrosis. Furthermore, similar CNP effects were observed in transgenic mice exhibiting fibroblast-specific CNP overexpression. In cultured-lung fibroblasts, CNP treatment attenuated TGF-ß-induced phosphorylation of Smad2 and increased mRNA and protein expression of α-smooth muscle actin and SM22α, indicating that CNP suppresses fibroblast differentiation into myofibroblasts. Furthermore, human lung fibroblasts from patients with or without interstitial lung disease substantially expressed GC-B receptor mRNA. CONCLUSIONS: These data suggest that CNP ameliorates bleomycin-induced pulmonary fibrosis by suppressing TGF-ß signaling and myofibroblastic differentiation in lung fibroblasts. Therefore, we propose consideration of CNP for clinical application to pulmonary fibrosis treatment.

C-type natriuretic peptide in combination with sildenafil attenuates proliferation of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is a malignant mesenchymal tumor and the most common soft tissue sarcoma in children. Because of several complications associated with intensive multimodal therapies, including growth disturbance and secondary cancer, novel therapies with less toxicity are urgently needed. C-type natriuretic peptide (CNP), an endogenous peptide secreted by endothelial cells, exerts antiproliferative effects in multiple types of mesenchymal cells. Therefore, we investigated whether CNP attenuates proliferation of RMS cells. We examined RMS patient samples and RMS cell lines. All RMS clinical samples expressed higher levels of guanylyl cyclase B (GC-B), the specific receptor for CNP, than RMS cell lines. GC-B expression in RMS cells decreased with the number of passages in vitro. Therefore, GC-B stable expression lines were established to mimic clinical samples. CNP increased cyclic guanosine monophosphate (cGMP) levels in RMS cells in a dose-dependent manner, demonstrating the biological activity of CNP. However, because cGMP is quickly degraded by phosphodiesterases (PDEs), the selective PDE5 inhibitor sildenafil was added to inhibit its degradation. In vitro, CNP, and sildenafil synergistically inhibited proliferation of RMS cells stably expressing GC-B and decreased Raf-1, Mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK) phosphorylation. These results suggested that CNP in combination with sildenafil exerts antiproliferative effects on RMS cells by inhibiting the Raf/MEK/ERK pathway. This regimen exerted synergistic effects on tumor growth inhibition without severe adverse effects in vivo such as body weight loss. Thus, CNP in combination with sildenafil represents a promising new therapeutic approach against RMS.

Cardiovascular and renal effect of CNAAC: An innovatively designed natriuretic peptide.

Natriuretic peptides (NPs) have natriuretic, diuretic and vasodilator effects. An innovative natriuretic peptide analogue called CNAAC (a new chimera peptide combining the C-terminus and ring of ANP with the N-terminus of CNP) was designed to determine whether it has any cardiovascular and renal effect. Abdominal aorta of rats were isolated and vascular ring perfusion was employed to compare the vasodilator effect and cGMP excretion effect of CNAAC with natural NPs. Urine volume and urine cGMP levels after intravenous injection of CNAAC and natural NPs were determined. Hemodynamic methods were employed to assess the effect of CNAAC and natural NPs on MAP. CNAAC relaxed abdominal aorta in a dose-dependent manner and was independent of endothelium. The vasodilating effect of CNAAC was significantly attenuated in the presence of NPR-A antibody, GC inhibitor, and KATP inhibitor and was abolished by PKG inhibitor. Abdominal aortic cGMP production increased after incubation with NPs. Urine volume, plasma cGMP, and urine cGMP increased and MAP decreased dramatically after intravenous injection of CNAAC. CNAAC has a potent vasodilating effect, probably by activating K(+) channels via NPR-A/sGC/cGMP pathway. Exogenous administration of CNAAC elicits diuretic and hypotensive effects.

Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1ß in cartilage explants.

C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1ß (IL-1ß). SEM showed uniform, 2 to 3 µm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1ß and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1ß in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

Exogenous C-type natriuretic peptide infusion ameliorates unilateral ureteral obstruction-induced tubulointerstitial fibrosis in rats.

Although many experimental therapeutic roles for C-type natriuretic peptide (CNP) have been documented in the field of cardiovascular and pulmonary-vascular disease, the therapeutic uses of CNP to nephropathies are not as well documented. In this study, we established a rat model of unilateral ureteral obstruction (UUO) to observe the beneficial effects of CNP on tubulointerstitial fibrosis (TIF). In UUO rats, CNP administration induced a significant increase in plasma CNP levels, and caused a significant decrease in blood urea nitrogen and creatinine levels. In addition, CNP infusion also alleviated the pathological lesions and collagen IV accumulation in the obstructed kidneys through downregulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 expression. In conclusion, exogenous CNP infusion can ameliorate UUO-induced TIF in rats. However, the use of CNP as a therapeutic agent requires further evaluation before being considered for human TIF.

C-type natriuretic peptide ameliorates ischemia/reperfusion-induced acute kidney injury by inhibiting apoptosis and oxidative stress in rats.

AIMS: Although atrial natriuretic peptide has been shown to attenuate ischemia-reperfusion (IR)-induced kidney injury, the effect of natriuretic peptide receptor (NPR)-B activation on IR-induced acute kidney injury is not well documented. The purpose of the present study was to identify the effect of C-type natriuretic peptide (CNP), a selective activator of NPR-B, on the IR-induced acute kidney injury and its mechanisms involved. MAIN METHODS: Unilaterally nephrectomized rats were insulted by IR in their remnant kidney, and they were randomly divided into three groups: sham, vehicle+IR, and CNP+IR groups. CNP (0.2µg/kg/min) was administered intravenously at the start of a 45-min renal ischemia for 2h. Rats were then killed 24h after I/R, and the blood and tissue samples were collected to assess renal function, histology, TUNEL assay, and Western blot analysis of kidney Bax and Bcl-2 expressions. KEY FINDINGS: The levels of blood urea nitrogen and serum creatinine were significantly increased in rats after IR compared with vehicle-treated rats. IR elevated apoptosis, Bcl-2/Bax ratio, TUNEL positivity, oxidative stress parameters, malondialdehyde concentration, and superoxide dismutase activity. IR also induced epithelial desquamation of the proximal tubules and glomerular shrinkage. CNP significantly attenuated the IR-induced increase in BUN and serum creatinine. Furthermore, CNP restored the suppressed renal cyclic guanosine 3' 5'-monophosphate levels caused by IR insult. SIGNIFICANCE: Study findings suggest that CNP could ameliorate IR-induced acute kidney injury through inhibition of apoptotic and oxidative stress pathways, possibly through NPR-B-cGMP signaling.

[Clinical condition and therapy of bone diseases].

Skeletal dysplasia is the term which represents disorders including growth and differentiation of bone, cartilage and ligament. A lot of diseases are included, and new disorders have been added. However, the therapy of most bone diseases is less well-established. Achondroplasia, hypochondroplasia, and osteogenesis imperfecta are most frequent bone diseases. There is no curative treatment for these diseases, however, supportive therapies are available ; for example, growth-hormone therapy for achondroplasia and hypochondroplasia, and bisphosphonate therapy for osteogenesis imperfecta. In addition, enzyme replacement therapy for hypophosphatasia is now on clinical trial.